Many patients with a severe acute illness have overwhelming systemic inflammation causing extensive tissue injury and multiple organ dysfunction syndrome (MODS). MODS is common, associated with high mortality, short-term morbidity, and significant chronic illness. When it develops, artificial organ support in an intensive care unit (ICU) is usually required. Despite prompt treatment with antibiotics and supportive care, effective therapy is not available yet. Disease manifestation involves a major inflammatory response accompanied by hypotension, vascular hyporeactivity, and cardiac depression. The latter is reversible and probably mediated by the synergistic effects of inflammatory cytokines and nitric oxide. In contrast, other organs, such as lungs, kidneys, and liver may quickly fail. MODS occurring in ICU patients is frequently deadly. Therefore, early categorisation of patients with different prognoses is important, but proves difficult in the prior art. Another problem in the art is the absence of a timely and/or specific biomarker for disease outcome.
Severe sepsis [SS] is a common acute illness in intensive care units [ICU] with high mortality rates and chronic morbidity. When associated with cardiovascular failure [termed septic shock] mortality rate is very high [50-80%]. A problem in the art is the lack of availability of biomarkers (such as organ specific biomarkers) that enable early diagnosis of impending septic shock to accurately categorise patients for personalised and focused interventions. There are approximately 30,000 annual cases of SS in the UK and numbers are rising. An uncontrolled inflammatory response to infection causes damage to and subsequently failure of organs such as the lungs, heart and kidneys. The underlying mechanisms are unclear. Medical care of patients combines prompt treatment with antibiotics and supportive care and often requires artificial organ support provided in an ICU. However, despite over 200 sepsis randomised controlled trials since 1992, no effective therapies exist. Consequently, an estimated 200,000 US ICU patients die every year. Hence, there is a need in the art for specific biomarkers that allow clinicians to, in real time, risk-stratify, initiate appropriate therapies, and/or accurately track response to treatment.
No existing biomarker reliably predicts mortality or response to treatment. Bedside clinicians are therefore unable to reliably predict risk of death, or monitor biological response to treatment. While >100 different potential sepsis biomarkers have been assessed, none has sufficient specificity or sensitivity to be clinically helpful. Common examples include C-reactive protein (CRP) and procalcitonin (PCT). Neither reliably predicts mortality. Neither is associated with treatment response.
Inforzato et al (2010 The Journal of Biological Chemistry, Volume 285, page 17681 to 17692) report that Pentraxin (PTX3) forms an asymmetric octamer. Biophysical studies of PTX3 are disclosed. Both recombinant and native human PTX3 molecules are studied. It is taught that the protein is composed of eight identical protomer sub-units. The structure of the assembled octamer is described. The assembled octamer is considered to be an asymmetric structure, consisting of two differently sized lobe regions connected by a stalk region. The authors consider that the N-terminal domain of the protomer provides the main structural determinant underlying this quaternary organisation. It is taught that the PTX3 octamer contains two FGF2 binding sites, whereas it is the tetramers that act as the functional units in ligand recognition. Inforzato et al do not disclose any connection between the different oligomeric PTX3s and any medical condition. Inforzato et al is confined to structural/biophysical characterisation of PTX3.
Inforzato et al (2008 The Journal of Biological Chemistry, Volume 283, pages 10147 to 10161) report study of the structural characterisation of PTX3, and its role in cumulus matrix organisation. The authors used both native and denaturing PAGE analysis and concluded that PTX3 is mainly composed of covalently linked octamers. The authors also studied PTX3 using mass spectrometry as well as cysteine/serine site directed mutagenesis. As a result of their studies, the authors report that cysteine residues at positions 47, 49 and 103 in the N-terminal domain of PTX3 are important for stabilising the tetrameric form of PTX3. They further report that cysteines at positions 317 and 318 are involved in linking PTX3 tetramers to form PTX3 octamers. The authors go on to produce a refined model of PTX3 structure, taking into account intrachain disulphide bonds. The authors study mutated PTX3 in a system rescuing defective cumulus matrix organisation, which is connected to ovulation. The authors found that PTX3 tetramers exhibited wild type rescue activity, whereas mutant PTX3 in the form of dimers had impaired functionality. The authors concluded that PTX3 tetramers were the functional molecular units required for cumulus matrix organisation and stabilisation. There is no teaching in this document regarding the importance of PTX3 octamers.
Rady (2007 Journal of Intensive Care Medicine, Volume 22, pages 386 to 388) presents an editorial article reviewing the field of sepsis and multi-organ dysfunction. In particular, the author discusses the cause-effect relationship of biomarkers in sepsis. In more detail, the author presents a detailed analysis of the field and study of B-type natriuretic peptide (BNP), and its use in sepsis and as a predictor of clinical outcome. BNP is a dominant marker in the field of sepsis. This document does not deal with PTX3. The author concludes that the cause-effect relationship of biomarkers is sepsis and clinical outcome remains elusive.
Further research regarding BNP and its correlation to disease outcome is presented by Rivers et al (2007 Journal of Intensive Care Medicine Volume 22, pages 363 to 373). The authors present a detailed study of the value of BNP, including its diagnostic, therapeutic and prognostic utility in critically ill patients. The authors present time course studies tracking BNP levels in patients with severe sepsis and septic shock. The authors draw robust conclusions about the value of BNP as a marker in this setting. Statistically significant associations are reported for BNP levels and organ dysfunction, myocardial dysfunction, global tissue hypoxia and mortality. Thus, the authors teach BNP as a useful and reliable marker in the early detection, stratification, treatment and prognostication of patients at high risk. This study does not discuss PTX3.
Pierrakos and Vincent (2010 Critical Care Volume 14, pages 1 to 18) present a literature review of biomarkers in sepsis. The authors searched scientific literature databases using various keywords. Their searching covered 3370 references involving 178 different biomarkers. The authors discuss the fact that many biomarkers have been evaluated for use in sepsis. They also remark that most of the biomarkers had been tested clinically, but relatively few had been used for diagnosis. PTX3 (Pentraxin 3) is mentioned in table to, line 26 of this document as being distinguished between septic shock and SIRS. None of the biomarkers reviewed had sufficient specificity or sensitivity to be employed in clinical practice. There is no disclosure in Pierrakos and Vincent of any of the multimeric states of PTX3.
US 2010/0292131 discloses kits and methods for the diagnosis, prognosis and prediction of sepsis in a subject, or for the differentiation between sepsis and SIRS in a subject. The disclosure is focussed on measuring the levels of pro-hepcidin (pro-HEPC) and/or a measurement of certain histone proteins. Pentraxin 3 is mentioned in this document as one of a panel of further biomarkers, which can be assayed simultaneously with the main markers being studied. This document does not appear to disclose information regarding the multimeric states of PTX3.
EP 2 092 342 B1 discloses a method for measuring plasma levels of pentraxin (PTX3). In particular, this document discloses a method for measuring PTX3 levels in plasma samples, comprising treating the sample with an agglutinating agent before determining the levels of PTX3. There is no disclosure or discussion in this document of PTX3 multimers.
Muller et al (2001 Critical Care Medicine Volume 29, pages 1404 to 1407) discloses how circulating levels of the long pentraxin PTX3 correlate with severity of infection in critically ill patients. The authors compare PTX3 with the more established marker C-reactive protein (CRP) in critically ill patients. The authors conclude that PTX3 is elevated in critically ill patients and correlates with severity of disease and infection. The authors do not deal with the multimeric forms of PTX3 in this disclosure.
Mauri et al (2010 Intensive Care Medicine Volume 36, pages 621 to 629) disclose persisting high levels of plasma PTX3 over the first days after severe sepsis and septic shock onset are associated with mortality. Ninety patients were studied. Patients were enrolled into the study on admission into intensive care. PTX3 levels were measured at various intervals.
Mortality was recorded at ninety days. The authors concluded that persisting high levels of circulating PTX3 over the first days from sepsis onset may be associated with mortality. They also concluded that PTX3 correlates with severity of sepsis and with sepsis associated coagulation/fibrinolysis dysfunction. The authors did not consider or assess PTX3 multimerisation.
Vänskä et al (2011 Haematologica Volume 96, pages 1385 to 1389) disclose that high Pentraxin 3 level predicts septic shock and bacteremia at the onset of febrile neutropenia after intensive chemotherapy of haematologic patients. The authors measured PTX3 and CRP in their study. The authors concluded that PTX3 is an early predictor of complications in haematologic patients with neutropenic fever. They also concluded that high PTX3 predicts septic shock and bacteremia already at the onset of febrile neutropenia. The authors did not examine the multimeric state of PTX3, but only assessed total PTX3 levels.
Daigo et al (2012 Molecular and Cellular Proteomics Volume 11, pages 1 to 12) disclose that the proteomic profile of circulating pentraxin 3 complex in sepsis demonstrates the interaction with azurocidin 1 and other components of the neutrophil extracellular trap. The authors used shotgun proteomics of circulating PTX3 complexes in order to study PTX3 ligands. The authors identified 104 candidate proteins, including various known PTX3 interacting proteins. The authors went on to show direct interaction of bactericidal proteins such as azurocidin 1 and myeloperoxidase with PTX3. Discussion of PTX3 oligomerisation appears to be confined to testing whether or not AZU1 binding required the known PTX3 N-terminal domain oligomer, as is the case for FGF2-PTX3 interaction. The authors conclude that AZU1-PTX3 binding is by a mechanism similar to PTX3-FGF2 binding. There is no study of PTX3 oligomers themselves in this publication.
Uusitalo-Seppälä et al (2013 Plos One Volume 8, pages E53661) disclose that pentraxin 3 is associated with severe sepsis and fatal disease in emergency room patients with suspected infection. The authors measured plasma PTX3 levels using commercially available solid-phase ELISA test on admission to emergency rooms. The authors compared PTX3 levels to CRP levels and PCT levels. The authors found that a high PTX3 concentration predicted severe disease and poor outcome. There is no discussion of, nor study of, PTX3 oligomerisation in this report.
The present invention seeks to overcome the problems associated with the prior art.